The ability to understand the effects of abused drugs on the central nervous system is critically dependent on understanding the chemical balance between neurons. Ideally, a chemical analysis technique must be capable of probing variations in the chemical profile within the cell (cytoplasmic analysis) as well as from well-defined loci in the extracellular compartments such as the synaptic cleft. The two main obstacles to achieving this goal with traditional analysis schemes have been: (l) the inability to handle ultrasmall sample volumes (on the order of 10(-15) to 10(-12) L), and (2) the inability to sensitively detect biomolecules that lack strong chromophores or easily oxidized groups. Falling into this category are peptides, for example, many of which are neurotransmitters and neuromodulators, as well as acetylcholine and glutamate. We have overcome these obstacles by coupling capillary electrophoresis a miniaturized highly efficient separation technique that handles sub-picoliter sample volumes, with a living cell biosensor that can detect virtually any neuroactive compound at the single-molecule level. The system uses ligand- receptor binding and signal-transduction pathways to amplify the presence of an analyte after electrophoretic separation. The transduced signal is measured using two approaches: (1) fluorescence microscopy that images changes in intracellular free Ca2+ concentrations in PC12 and N6108-15 cultured cell lines using fluo-3, a dye that increases its fluorescence quantum yield severalfold upon binding with Ca2+, and (2) measurement of transmembrane currents in Xenopus laevis oocytes microinjected with mRNA that encodes a specific receptor. The strength of these biosensor systems over conventional analysis schemes is the ability to detect, with unsurpassed selectivity, a fractionated molecule in its native state. With this technique, we have been able to detect bradykinin and acetylcholine in complex biological mixtures. Further, by using selective antagonists to the activated receptor, we have shown that it is possible to assign unambiguously a band separated by capillary electrophoresis as an agonist to that receptor and also to deduce whether the agonist solely operates via one receptor subclass. It is our belief that this methodology can aid in the development of models explaining how drugs (or endogenous substances whose effects are coupled through drugs) disrupt normal neurochemical communication and affect neuronal plasticity. The long-term objective of this research program is to implement capillary electrophoresis coupled to single-cell biosensors to probe the chemical connectivity between individual neurons and to identify novel neurotransmitters.